Production of replicative hepatitis C virus

ABSTRACT

A nucleic acid having a first nucleotide sequence encoding an infectious hepatitis C virus, a second nucleotide sequence encoding a ribozyme, and an inducible promoter operably linked to the first and second nucleotide sequences, the ribozyme being configured to remove a 3′ sequence unnecessary for replication of the infectious hepatitis C virus from a transcript initiated by the inducible, is described. A cell containing the nucleic acid and methods of using the cell are also described.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under NationalInstitutes of Health grants RO1 DK57857-01 and RO1 AI43478-02. TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to virology and antiviral drug screening.

BACKGROUND OF THE INVENTION

The development of anti-viral strategies against hepatitis C virus (HCV)infection has been hindered by the lack of an ideal animal model, oreven a cell culture system, for HCV replication. One characteristic ofan ideal HCV animal model or cell culture system would be the ability toinduce expression of an infectious HCV in a cell in vitro or in vivo.

The HCV genome consists of a positive strand RNA that encodes a singleprecursor viral protein that is cleaved by cellular and viral proteasesto generate viral structural and non-structural proteins, respectively.Non-translated regions 5′ and 3′ (5′NTR and 3′NTR) to the open readingframe encoding the precursor protein are also involved in viralreplication. For a review of HCV, see Houghton, “Chapter 32, Hepatitis CViruses,” in: Fields Virology, 3rd ed., Fields et al. eds., pp1035-1058, 1996, Lippincott-Raven Publishers, Philadelphia, Pa.

Infection with HCV is one of the leading causes of chronic liver diseasethroughout the world. Chronic infection nearly always ensues after acuteexposure to HCV, and chronically infected individuals develop cirrhosisand hepatocellular carcinoma at a dramatically elevated rate comparedwith the rate of disease in an uninfected population.

The development of more effective treatments has been limited by thelack of an effective tissue culture or small animal model of infection.HCV replication systems based on a self-replicating HCV RNA replicon isdependent only on expression of the nonstructural viral proteins.Therefore, such systems do not recapitulate all steps of the HCV viruslife cycle, some of which may be effective targets for antiviralintervention. Cell lines stably transfected with the HCV precursor arenot capable of HCV RNA replication. Consequently, such cell lines cannotbe used to screen for antiviral drugs that block viral RNA replication.RNA transcripts from an infectious HCV cDNA clone can replicate inchimpanzees, but any model that requires the use of large, expensiveprimates is impractical.

SUMMARY OF THE INVENTION

To address the historical and intractable limitations in the above HCVreplication models, the invention provides an inducible system forproducing infectious or replicative HCV, as described in the Examplebelow, thereby fulfilling a long-felt need for an ideal HCV replicationsystem.

The invention is based on the development and implementation of aninducible cell-based system for producing replicative HCV. Because theHCV produced by this system is replicative and has the full complementof genetic material found in HCV (e.g., naturally occurring HCV), allaspects of the HCV life cycle can be examined, e.g., in screening assaysfor candidate antiviral compounds. The inducibility of the system allowsthe propagation of cells or animals containing HCV genetic materialwithout the damaging effects of HCV replication. Thus, viral replicationis induced only when desired, for example, in a particular step thatrequires infectious HCV replication in an assay.

Accordingly, the invention features a nucleic acid having a firstnucleotide sequence encoding an infectious hepatitis C virus, a secondnucleotide sequence encoding a ribozyme (e.g., a hepatitis D virusribozyme), an inducible promoter (e.g., a T7 promoter) operably linkedto the first and second nucleotide sequences, and optionally atranscription termination signal (e.g., a T7 transcription terminationsignal) operably linked to the first and second nucleotide sequences,the ribozyme being configured to remove a 3′ sequence unnecessary forreplication of the infectious hepatitis C virus from a transcriptinitiated by the inducible promoter and optionally terminated by thetranscription termination signal. The invention also includes cells thatharbor a nucleic acid of the invention.

A method of producing infectious HCV is also included in the invention.A cell containing the nucleic acid of the invention (e.g., as an episomeor an integrated cassette) can then be used to generate infectious HCVby inducing the inducible promoter of the nucleic acid. For example, ifthe promoter is a T7 bacteriophage promoter, HCV is produced byexpressing a T7 RNA polymerase in the cell. The T7 RNA polymerase inturn can be expressed by infecting the cell with a viral vector (e.g., avaccinia vector) encoding the T7 RNA polymerase. Alternatively, the cellcan contain an episomal plasmid or genomic transgene (e.g., delivered bya retrovirus) that expresses T7 RNA polymerase. Regardless of thevectors used to express T7 RNA polymerase, the expression of thepolymerase can itself be regulated, depending on the genetic elementsoperably linked to the sequence encoding the polymerase.

The invention further includes a screening method for identifying acompound (e.g., a polypeptide, small molecule, or nucleic acid, such asan antisense nucleic acid or ribozyme) that inhibits replication of anHCV. The method includes (1) providing a test cell containing a nucleicacid of the invention, (2) inducing the inducible promoter of thenucleic acid, (3) contacting the test cell with a candidate compound,and (3) detecting a decrease in the amount of infectious hepatitis Cvirus produced by the test cell compared to the amount of the infectioushepatitis C virus produced by a control cell. The detecting step caninclude measuring (e.g., by PCR) the amount of negative strand hepatitisC viral RNA in the cell or the amount of positive strand hepatitis Cviral RNA in the cell or in cell-free virions produced by the cell.

Since it is possible that the candidate compound inhibits HCVreplication by inhibiting a viral or cellular protease responsible forcleaving the HCV precursor protein, the screening method can furtherinclude determining whether a hepatitis C virus structural ornon-structural protein is cleaved from a hepatitis C virus precursorprotein in the cell after the contacting step, e.g., by protein gelelectrophoresis.

As used herein, “inhibits” or “inhibition” means any measurable decrease(e.g., 10%, 20%, 50%, 90%, or 100%) in an activity of interest.

As used herein, an “infectious hepatitis C virus” means an HCV that iscapable of propagation in a population of cells in vivo or in vitro.Therefore, an infectious hepatitis C virus minimally contains (1) asequence encoding a precursor protein and (2) 5′ and 3′ non-translatedflanking sequences sufficient to support virus replication (i.e., eachstep of the virus life cycle) in a cell population.

By one genetic element being “operably linked” to another is meant thata genetic element (either in a plus strand, minus strand, or doublestranded form) is structurally configured to operate or affect anothergenetic element. For example, a promoter operably linked to a sequenceencoding a polypeptide means that the promoter initiates transcriptionof a nucleic acid encoding the polypeptide, and a transcriptiontermination signal operably linked to the sequence encoding thepolypeptide means that the transcription termination signal terminatestranscription of a nucleic acid encoding the polypeptide.

The nucleic acids and methods of the invention provide a HCV replicationsystem amenable to comprehensive, yet relatively inexpensive (ascompared to infection of a primate), antiviral drug screening methods.Because the HCV replication system performs all steps of the virus lifecycle, candidate antiviral compounds can be screened for activityagainst any vital viral or cellular drug target involved in virusreplication. In addition, the HCV replication system is inducible,thereby allowing cells to vigorously replicate in the absence of HCVuntil virus replication becomes necessary for the particular step of ascreening assay performed. Thus, the nucleic acids and methods of theinvention remove a substantial obstacle in anti-HCV drug development.

Other features or advantages of the present invention will be apparentfrom the following detailed description, and also from the claims.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic diagram of the infectious HCV production systemdescribed herein. “8” is the hepatitis D ribozyme. “T7” above the box,on the left side of the schematic diagram for “DNA” is the T7 promoter.“T7” above the box, on the right side of the schematic diagram for “DNA”is the T7 transcription termination signal. “vvT7” is the vaccinia virusvector encoding the T7 RNA polymerase.

FIG. 2 is a schematic diagram of a non-infectious control DNA constructand of the positive strand RNA produced from it.

DETAILED DESCRIPTION

The nucleic acid, such as an expression plasmid, of the invention can beconstructed using standard methods and reagents in the art of molecularbiology. For example, inducible promoters, such as the T7 promotersystem (Aoki et al., Virology 250:140-150, 1998;

-   -   WO 98/39031) can be used to provide controlled expression of the        infectious HCV clone. Alternatively, a tetracycline-inducible        promoter system can be used (Moradpour et al., Hepatology        28:192-201, 1998). The inducible promoter is then operably        linked to an infectious clone of an HCV, such as the one        described in Yanagi et al., Proc. Natl. Acad. Sci. USA        94:8738-8743, 1997; or Kolykhalov et al., Science        277:570-574, 1997. At the 3′ end of the HCV clone, a ribozyme        (e.g., a hepatitis D virus ribozyme) and optionally a        transcription termination signal (e.g., a T7 transcription        termination signal) is attached. Ideally, a cis-acting ribozyme,        such as a HDV ribozyme, that cleaves at the boundary with the 3′        terminus of the HCV RNA is suitable for this purpose. After the        HCV clone is transcribed, the ribozyme serves to remove itself        and other 3′ sequences which may hinder virus replication.

Depending on the inducible promoter used to drive transcription of theinfectious HCV clone, the method for inducing HCV production will vary.For example, if a T7 promoter is used, then transcription is induced byexpressing a T7 RNA polymerase in a cell harboring the nucleic acid(e.g., as a result of transfection, viral vector delivery, or genomicDNA integration). The polymerase can be expressed using a viral vector,such as the vaccinia vector described in the Example below, or theadenovirus vector described in Aoki et al., supra. Alternatively, a T7polymerase expression vector under the control of a mammalian promotercan be introduced into the cell to induce HCV production. In addition,the cell can already contain a stably integrated expression cassettethat is induced to express T7 polymerase, thereby producing replicativeHCV.

To allow propagation of HCV, induced cells are optionally mixed with apopulation of cells that do not produce HCV but are permissive for HCVinfection. A mixed or unmixed population can then be used in screeningassays for candidate antiviral compounds. Depending on the nature of acandidate compound, contacting the population of cells with the compoundcan involve a variety of standard techniques. If the candidate compoundis a small molecule drug that can pass through lipid membranes, all thatis needed for the contacting step is the addition of the compound to anaqueous mixture containing the cells. However, if the compound is aprotein or nucleic acid intended to elicit its antiviral effect inside acell, the contacting step may require other techniques. A proteincompound can be delivered into the cell by encapsulation in liposomes orfusion to a viral protein that is delivered inside a cell by viralinfection. A nucleic acid can be delivered into the cell using a viralvector or a transfection protocol (e.g., electroporation). Methods ofintroducing proteins and nucleic acids into a cell are well known in theart of molecular biology.

After the contacting step, the amount of infectious hepatitis C virusproduced in the presence of the compound is measured and compared to acontrol amount of infectious hepatitis C virus. The control amount isgenerally an amount observed in a similar population of cells grown inthe absence of the candidate compound.

The amount of HCV can be measured using any suitable direct or indirectmethod. Several suitable methods are known in the art. For example, theamount of positive strand viral RNA in cells and in virions can bemeasured using quantitative PCR. The amount of minus strand RNA can alsobe measured using PCR. In addition, viral proteins, rather than viralnucleic acids, can be measured, e.g., in the supernatant of anHCV-infected cell culture, using an enzyme linked immunosorbent assay(ELISA). Such assays typically utilize monoclonal or polyclonalantibodies to capture viral antigens in a sample. The captured antigensare then detected using labeled antibodies that specifically bind to thecaptured antigen at an epitope different from the one to which thecapture antibody binds. HCV enzyme-linked immunoassays are availablefrom Abbott Laboratories. In addition, any decrease in the level ofcleavage of the viral precursor protein caused by the presence of thecandidate compound provides information regarding the mode of action ofthe compound. For example, decreased cleavage could lead to askingwhether the compound binds and inhibits a viral or cellular protease.Thus, an examination of the level of precursor protein cleavage can be avaluable step in the screening methods of the invention.

The methods of the invention can be performed repetitively and inparallel to screen libraries of compounds (e.g., a small moleculelibrary, a peptide library, or a single chain antibody library) forcandidate antiviral drugs. For example, cells having a nucleic acid ofthe invention can be cultured in the wells of a 96-well microtiterplate. Before or after induction of HCV replication, each member of thecompound library is deposited into one well, and the level of HCVreplication determined in the well. Thus, automation of the methods ofthe invention are especially amenable to high throughput screening ofchemical libraries.

This invention can also be used to identify key cellular proteinsinvolved in HCV RNA replication by identifying replication complexes,and to identify host cellular genes that are induced by expression ofreplicative HCV.

One skilled in the art can, based on the above disclosure and theexample described below, utilize the present invention to its fullestextent. The following example is to be construed as merely illustrativeof how one skilled in the art can make and use the inducible HCVreplication system described herein. Any publications cited in thisdisclosure are hereby incorporated by reference.

EXAMPLE

Materials and Methods

Preparation of constructs. The HCV construct originally used to generateinfectious transcripts was the H77 clone (Yanagi et al., Proc. Natl.Acad. Sci. USA 94:8738-8743, 1997). The plasmid containing the clone,pCV-H77, contains the full-length genotype 1a HCV sequence of strainH77. The plasmid contains a T7 promoter immediately upstream of theviral cDNA and was adapted at its immediate 3′ terminus with thehepatitis delta virus cis-acting ribozyme (Wang et al., Nature323:508-514, 1986) in continuity with the T7 terminator sequence. Toaccomplish this, a synthetic antisense oligonucleotide was made, theoligonucleotide being the complement of the following contiguoussequences: the terminal 3′ 20 nucleotides of the H77 strain, followedimmediately by the 85-nucleotide HDV ribozyme, followed immediately bythe 48-nucleotide T7 terminator. This 153-nucleotide sequence wasdivided into two overlapping 85-nucleotide primers, each sharing 17nucleotides in common. These primers were synthesized (IDT, Coralville,Iowa) and used as the downstream primers in a series of PCR reactions.

A sense oligonucleotide of 85 nucleotides in length, corresponding toHCV H77 sequences 9367-9451, was also synthesized in a similar manner.H77 numberings, as use herein, are as designated in Yanagi et al.,supra. The sense primer and the inner antisense primer were used in aPCR reaction with 10 ng of pCV-H77 as a template under the followingconditions: 50 pmol of primer, 0.5 U Taq polymerase, 1.5 mM MgCl₂, 0.5μM each dNTP. The 100 μl reaction was carried out with 20 cycles of PCRunder the following cycling conditions: 95° C. for 1 minute, 65° C. for1 minute, and 72° C. for 1 minute. The PCR product was gel purified(QIAquick, QIAgen, Chatsworth, Calif.) and cloned into the vectorpcDNA3.1 V5/His-TOPO (Invitrogen, Carlsbad, Calif.). This product thenserved as the template for a second PCR reaction under identicalconditions using the sense primer and the outer antisense primer. Theproduct of the second amplification was again cloned intopcDNA3.11N5/His-TOPO to generate pHCV-Rz-TOPO. The sequence of thisproduct was confirmed bi-directionally by dideoxy sequencing.

Because the parent plasmid for pCV-H77C, pGEM-9z, contained a second T7promoter at its original multiple cloning site just downstream of theHCV cDNA, this sequence was removed by excising the XbaI-SfiI fragment.A synthetic XbaI-MluI-MluI-SfiI linker pair was generated, and ligatedinto the XbaI-SfiI-digested pCV-H77C. Successful insertion of a singlelinker was confirmed by sequencing. This product, pCV-H77C-Mlu, wasdigested with AflII and MluI, and the AflII-Mlul fragment isolated frompHCV-Rz-TOPO was subcloned into this vector, to yield pT7-flHCV-Rz.Successful insertion of HDV Rz and T7 terminator sequences was confirmedby bi-directional sequencing.

To confirm the dependence of the replication system on intact HCVnon-structural proteins, which had been postulated to be essential forRNA replication, a deletion mutant was produced by removing theBglII-BglII fragment from pT7-flHCV-Rz (FIG. 2). The deletion spannedHCV nucleotides 3237-8939. This deletion removed the downstream portionof NS2 to NS5B, inclusive of the active site motif of the NS5BRNA-dependent RNA polymerase. The 5.7 kb deletion was predicted to keepthe polyprotein in frame. The deletion was confirmed by sequencing ofpT7-HCVD BglII-Rz. For experiments designed to examine vaccinia-T7efficiency, the positive control plasmid OS8 contained a T7 promoterflanking the β-galactosidase gene.

Cell lines. Cells were maintained in DMEM (BRL Gibco, Rockville, Md.)containing penicillin (50 IU/mL) and streptomycin (50 μg/mL) and wassupplemented with 10% fetal calf serum. CV-1 cells were obtained fromthe American Type Culture Collection. For some experiments, HepG2 andHuh7 cell lines were used for transfection/infection.

Transfection/infection experiments. Expression of HCV was carried outusing an adaptation of the binary expression system described in Fuerstet al., Proc. Natl. Acad. Sci. USA 83:8122-8126, 1986. Subconfluent CV-1cells, chosen because of their enhanced transfection efficiency, weretransfected in 6-cm tissue culture plates using 1 μg of plasmid(pT7-flHCV-Rz, pT7-HCVDBglII-Rz, OS8) with 3 μl of Lipofectaminetransfection reagent (GIBCO BRL) in serum-free DMEM. Following 4 hoursof transfection, the cells were washed and replaced with DMEM containing10% FCS. Twenty-four hours post-transfection, recombinant vaccinia-T7polymerase (vTF7-3; Fuerst et al., supra) was added to the cells at anMOI of 10. Twenty-four hours post-vTF7-3 infection, cells were lysed andanalyzed for RNA and protein expression. The positive control vector OS8verified successful T7 polymerase expression.

Strand-specific RT-PCR. To confirm successful HCV RNA strand synthesis,strand-specific RT-PCR was carried out on lysates oftransfected/infected cells. RNA was extracted using TRIzol reagent(GIBCO BRL), and subjected to two rounds of DNase I digestion (5 U,Boehringer Mannheim, Indianapolis, Ind.) at 37° C. for 60 minutes. TheRNA was then phenol/chloroform-extracted and resuspended in DEPC-treatedwater. The purified RNA was then subjected to strand-specific RT-PCRusing primers corresponding to the 5′ and 3′ genomic and antigenomic H77RNA. The following oligonucleotide sequences, all 20-mers, were used: 5′end sense, H77 nt 29-48; 5′ end antisense, nt 390-371; 3′ end sense, nt9241-9260; and 3′ end antisense, nt 9361-9342. For detection of genomic(+) strand RNA, the antisense primer was used for the reversetranscription step. For detection of antigenomic (−) strand RNA, thesense primer was used for the reverse transcription step. Reversetranscription was carried out in 20 μl reactions with the followingcomponents: 1 μl RNA, 25 pmol RT primer, 0.5 U AMV reverse transcriptase(Perkin Elmer, Branchburg, N.J.), 1.5 mM MgCl₂, 0.5 μM each dNTP, and 1URNasin (Perkin Elmer). The reaction was carried out at 42° C. for 15minutes, and the enzyme was heat inactivated for 10 minutes at 99° C.The resulting product was treated with three successive rounds of DNaseI (Boehringer Mannheim) at 37° C. for 30 minutes each, and the finalproduct was purified by phenol/chloroform extraction. The cDNA was thensubjected to 25 cycles of PCR using 25 pmol each of the relevant senseand antisense primers, 0.5 μM each dNTP, 1.5 mM MgCl₂, and 0.5 U Taqpolymerase. Reaction products were then analyzed by 1.6% agarose gelelectrophoresis.

Ribonuclease protection assay (RPA). To confirm HCV replicative RNAsynthesis by a second line of investigation, RPA was performed on theextracted RNAs. A probe was used for specific detection of antigenomicHCV RNA at the 3′ terminus of the genome. To accomplish this, the vectorpHCV-3′T (Chung et al., Biochem. Biophys. Res. Commun. 254:351-362,1999) was used to generate a sense probe corresponding to the terminalHCV RNA. This vector contains the highly conserved 98-nucleotide 3′terminal sequence (also conserved in H77) that had been adapted andcloned into the EcoRI and XbaI sites of pSP72. Following linearizationof pHCV-3′T by XbaI, an α-³²P-UTP-labeled probe was generated by invitro transcription using T7 polymerase according to manufacturer'sprotocol (Ambion, Houston, Tex.). This probe was purified byphenol/chloroform extraction and then used for RPA. For all RPAs, theRPA II kit was used in accordance to manufacturer's instructions(Ambion). RPA products were separated by 8 M urea/5% PAGE. For the HCVreplicative strand, the expected protected fragment size was 210nucleotides. As a positive control, hybridization was performed with thepT7-flHCV-Rz DNA.

To generate RPA probes for human β-actin and GAPDH, commerciallyavailable antisense control DNA templates pTR1-β-actin and pTR1-GAPDH(Ambion) were used. As with the HCV template, in vitro transcription inthe presence of α-³²P-UTP was carried out using T7 RNA polymerase(MAXIscript, Ambion). The probes were phenol/chloroform extracted,purified, and used in RPA experiments conducted according to themanufacturer's instructions (RPA II, Ambion). To generate an antisenseRPA probe for the detection of β-galactosidase mRNA, a PCR approach wasused in which the antisense primer was adapted upstream with the T7promoter sequence. Oligonucleotide primers corresponding to the codingsequence of β-galactosidase in the vector OS8 were synthesized asfollows. The sense strand was 5′-CCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTG-3′(SEQ ID NO:1), and the antisense was 5′-TATACGACTCACTATAGGCCATTCGCCATTCAGG-3′ (SEQ ID NO:2; T7 promoter sequence underlined).

PCR was carried out using 10 ng of OS8 as template under the followingconditions: 25 pmol each primer, 0.5 μM each dNTP, 1.5 mM MgCl₂, 0.5 UTaq polymerase. Twenty-five cycles were performed under the followingconditions: 95° C. for 1 minute, 45° C. for 1 minute, 72° C. for 1minute. PCR products were separated by agarose gel electrophoresis andgel purified (QIAquick, QIAgen). The purified template DNA was then usedfor generation of α-³²P-UTP-labeled antisense probe by in vitrotranscription with T7 polymerase (MAXIscript, Ambion). RPA was carriedout as described above. For β-actin, GAPDH, and β-galactosidase probes,hybridization was carried out using the original template DNAs aspositive controls. For the ribavirin and amantadine studies, asimultaneous RPA using GAPDH probe with HCV (−) strand probe wasperformed, and the results displayed on the same gel.

Western immunoblotting. Lysates were extracted using Laemmli 2×SDSsample-extraction buffer (Sigma, St. Louis, Mo.). Total protein wasquantitated by the Bradford assay. Equal quantities of protein wereloaded and separated by 10% SDS-PAGE. Following transfer of the gels toPVDF membranes, Western immunoblotting was carried out using theECL-Western detection method (Amersham, Piscataway, N.J.). For HCVprotein detection, polyclonal antisera were used. The antisera werepooled from HCV-infected patients whose sera were reactive by therecombinant immunoblot assay (RIBA-2, Abbott Labs, Chicago, Ill.) at adilution of 1:50. Horseradish peroxidase-conjugated rabbit anti-humanantibody (Amersham) was used at 1:5000 for detection. Five micrograms ofrecombinant HCV core protein (Austral Biologicals, San Ramon, Calif.)was used as a positive control. For β-galactosidase detection,monoclonal anti-β-galactosidase (Promega, Madison, Wis.) was used as theprimary antibody at a dilution of 1:5000. For β-actin, monoclonalanti-actin antibody (Chemicon, Temecula, Calif.) was used at a dilutionof 1:200. For both actin and galactosidase immunoblots, conjugatedrabbit anti-mouse (Amersham) antibody at 1:10,000 dilution was used assecondary antibody.

Antiviral inhibitor studies. Purified, recombinant IFN-α-2b (INTRON A,Schering Plough Research Institute, Kenilworth, N.J.) was used atincremental doses of 0, 2000, 6000, or 15,000 units/plate. The drug wasadded to the media at the conclusion of transfection. Time courseexperiments indicated that IFN-α-2b's inhibition of vaccinia-T7polymerase was minimized when IFN-α-2b was given 20 hours prior to theintroduction of vTF7-3.

Ribavirin (Schering Plough Research Institute) was given at the doses of0, 10, 20, 80, 120, 160, and 200 mg/plate were added to the media 20hours prior to introduction of vTF7-3. These doses were comparable tothose achievable at physiologic concentrations and at the highest dosesgiven in excess of those found to cause cellular toxicity (Ilyin et al.,Hepatology 27:1687-1694, 1998). The results were unchanged whenribavirin was given 0 and 12 hours before vTF7-3 infection.

Amantadine-HCl (Sigma) was used at doses comparable to pharmacologicallyachievable concentrations (Shannon et al., Antimicrob. Agents Chemother.20:769-776, 1981). Doses of 0, 1, 10, and 100 μg/plate were added to themedia at 20 hours prior to vTF7-3 infection. Results seen were unchangedwhen amantadine was given 0 and 12 hours before vTF7-3 infection.

Results

Generation of a binary HCV expression system in mammalian cells. Toexplore whether the early steps in the HCV lifecycle could berecapitulated using a DNA-based approach, the plasmid pCV-H77 provided astarting point for the genetic manipulations. pCV-H77 contained afull-length genotype 1a sequence, inclusive of the highly conserved 5′and 3′ untranslated sequences. pCV-H77 was adapted at its 3′ terminuswith the cis-acting hepatitis delta ribozyme followed immediately by theT7 transcription termination sequence (FIG. 1). The final construct wasdesignated pT7-flHCV-Rz. Because the upstream end of the viral sequencelied immediately downstream of the T7 promoter, T7 polymerase wasexpected to generate, by transcription, a full-length viral RNA genomebearing bona fide termini for strand replication.

When CV-1 cells were transfected with pT7-flHCV-Rz and infected withrecombinant vaccinia encoding T7 polymerase (vTF7-3), detectable RNAcorresponding to the genomic or (+) strand and the antigenomic or (−)strand of the viral genome was detected. Strand-specific RNA synthesiswas detected by two lines of investigation: strand-specific RT-PCR andribonuclease protection assay. As confirmation of full antigenomicstrand synthesis, (−) strand RNA sequences corresponding to both the 5′and 3′ untranslated portions of the genome (i.e., the 5′ and 3′ terminiof the antigenomic strand) were detected by RT-PCR. Using an RNaseprotection assay, (−) strand RNA corresponding to the 3′ terminus of thegenome was also detected. This synthesis was dependent on transfectionwith HCV sequences (pT7flHCVRz) and infection with vTF7-3, but notdependent on pT7-flHCV-Rz alone or on vTF7-3 combined with an unrelatedtemplate. Thus, the replication system succeeded in producing the fullcomplement of viral protein synthesis.

To confirm successful viral protein synthesis, Western blotting wasperformed using polyclonal antisera directed against genotype 1 HCVinfection. Immunoreactive HCV core protein was expressed only in thepresence of T7-flHCV-Rz and vaccinia-T7, but not with either alone or inthe presence of vTF7-3 and an unrelated expression construct. Thesuccessful synthesis of β-galactosidase from a control vector confirmedthe validity of our binary expression system for CV-1 cells.

HCV antigenomic strand synthesis is dependent on full-length HCVsequences. To confirm that this specific RNA strand synthesis wasdependent on the expression of viral proteins, as was expected, theexperiments described above was repeated with an expression constructdeleted in the region from NS2-NS5 (FIG. 2). This deleted regioncontained the NS3 RNA helicase and the key catalytic domains of the NS5BRNA-dependent RNA polymerase. Despite inducible production of HCV coreprotein, in levels comparable to the full length construct, nodetectable (−) strand synthesis was seen by either RT-PCR or RNaseprotection assay.

In the case of the deletion mutant, the inability to carry outsuccessful (−) strand synthesis in the face of preserved structuralprotein synthesis and in the presence of an intact RNA template terminusstrongly suggested that synthesis of antigenomic RNA requires thepresence of HCV nonstructural proteins. The finding of equivalent levelsof core protein between the wild-type and mutant constructs suggestedthat the observed protein synthesis was most likely attributable to theinitial effects of polyprotein translation by host ribosomes rather thanthe result of repeated rounds of viral genome replication.

Interferon-α directly and selectively inhibits HCVRNA and proteinsynthesis. Having established a successful binary cell-based expressionsystem for (−) strand HCV RNA synthesis, the effects of potentiallyclinically active antiviral compounds on (−) strand synthesis was nextexamined. It was known that interferon-α-2b (IFN-α), with or withoutribavirin, was the only compound approved for the treatment of chronicHCV infection. Without a replication system for HCV, one could notseparate IFN-α's direct antiviral effects from its indirect effects,such as immunomodulatory effects. Therefore, the HCV replication systemdescribed herein was used to examine the direct effects of IFN-α on HCVreplication.

Recombinant interferon-α-2b was added to the cell culture system used togenerate replicative HCV, as described above, at doses predicted to havean antiviral effect in tissue culture. A dose-dependent inhibition ofHCV (−) RNA synthesis by IFN-α was found at doses from 2000 to 15,000units/well, which had no effect on RNA levels of actin andβ-galactosidase in the RNase protection assay. When IFN-α's effects onprotein synthesis were examined, a dose-responsive inhibition of HCVcore protein synthesis by IFN-α was observed. This effect wassubstantially greater than IFN-α's modest effects on β-galactosidaselevels. Actin protein synthesis was unaffected. Taken together, thesedata demonstrated that interferon-α exerted a direct antiviral effect onHCV RNA and protein synthesis.

Neither ribavirin nor amantadine inhibit HCVRNA synthesis. Thenucleoside analogue ribavirin was known to be an approved agent, inconjunction with interferon-α, for the treatment of chronic hepatitis Cinfection. While ribavirin did not appear to be effective against HCV inmonotherapy, the drug's antiviral effects could be augmented by IFN(Davis et al., N. Engl. J. Med. 339:1493-1499, 1998; and Hoofnagle etal., J. Hepatol. 31:264-268, 1999). To determine whether this compoundhad direct anti-HCV activity, ribavirin was tested at doses of 10-200mg/well. These doses are comparable to, and in excess of, those found atclinically applicable concentrations. No inhibitory effect of ribavirinon HCV (−) RNA synthesis was found.

In some reports, amantadine demonstrated an antiviral effect againstseveral RNA viruses, including HCV. When amantadine was tested at doses(1-100 μg/well) in excess of clinically relevant concentrations,amantadine showed no activity against HCV (−) strand synthesis.

Taken together, neither ribavirin nor amantadine demonstrated directactivity against HCV (−) RNA synthesis, suggesting that any observedeffect on the control of in vivo infection may be attributable to anindirect mode of action, e.g., an immunomodulatory mode of action.Further, when combined with the positive results obtained for IFN-αabove, the studies described herein demonstrate that the HCV replicationmodel can be used to determine whether candidate antiviral compoundshave a direct affect on viral replication.

1-16. (canceled)
 17. A nucleic acid comprising a first nucleotidesequence encoding an infectious hepatitis C virus, a second nucleotidesequence encoding a ribozyme and, an inducible promoter operably linkedto the first and second nucleotide sequences, the ribozyme beingconfigured to remove a 3′ sequence unnecessary for replication of theinfectious hepatitis C virus from a transcript initiated by theinducible promoter.
 18. The nucleic acid of claim 17, wherein theinducible promoter is a T7 promoter.
 19. The nucleic acid of claim 17,wherein the ribozyme is a hepatitis D virus ribozyme.
 20. (canceled) 21.A cell comprising the nucleic acid of claim
 17. 22. The nucleic acid ofclaim 17, wherein the nucleic acid is a vector.
 23. The nucleic acid ofclaim 22, wherein the vector is a plasmid.
 24. The nucleic acid of claim17, wherein the nucleic acid is an integration cassette.
 25. A cellcomprising the nucleic acid of claim
 22. 26. A cell comprising thenucleic acid of claim 24.